Apoptosis is a process of programmed cell death by which multicellular organisms selectively delete cells. The term necrosis is used to describe the morphological changes undergone by cells that die by processes other than apoptosis. Apoptosis is characterized by a progressive condensation of the chromatin to the inner face of the nuclear membrane, cell shrinkage with consequent loss of membrane contact with neighboring cells, and fragmentation of the cells with formation of membrane-bound acidophilic globules (apoptotic bodies).
The DNA of cells that have undergone apoptosis is cleaved into fragments that are multiples of approximately 180 base pairs. These fragments can be seen after agarose gel electrophoresis as a characteristic "ladder" develops. This ladder is widely used as a biochemical marker for discriminating apoptotic cell death from necrotic cell death as the DNA of necrotic cells is randomly degraded and does not produce a ladder. The ladder develops as a result of cleavage of nuclear DNA within the linker regions between nucleosomes (1,2). Double strand cleavage results from frequent nicks on both DNA strands (3).
Although the endonuclease responsible for the cleavage of DNA in apoptotic cells has not been definitively identified, candidate nucleases with properties consistent with their involvement in apoptosis have been identified in apoptotic cells (4-6).
The endonucleases that have been identified in apoptotic cells are generally similar in their properties to pancreatic DNase I (7). Specifically, these endonucleases share the following characteristics:
(i) the DNA ends produced by DNase I cleavage (5'-phosphate and 3'-hydroxyl) are the same as those found in apoptotic nuclei (8-10); PA1 (ii) DNase I-transfected COS cells show chromatin changes similar to those seen in apoptosis (11); and PA1 (iii) DNase I cleavage of chromatin produces the same characteristic nucleosomal DNA fragments that can be isolated from apoptotic cells (1).
Although DNase I has been detected in cells undergoing apoptosis and the tissue distribution of DNase I is consistent with a role in apoptosis (12, 13), the endonucleases partially purified from apoptotic cells were shown to be distinct from DNase I (4-6). There is less evidence for the involvement of DNase II and other endonucleases in apoptosis (14, 15).
Presently, the terminal deoxynucleotidyl transferase(TdT)-mediated biotinylated dUTP nick end labeling (TUNEL) method is used to detect apoptotic cells in tissue sections. The TUNEL method utilizes TdT to incorporate a biotinylated deoxyuridine label into DNA fragments containing a 3'-hydroxyl group. The label can be detected using a variety of avidin/streptavidin based detection methodologies. Although 3'-hydroxyl groups are present in the double stranded DNA breaks in the apoptotic cells, they are also present in the DNA of cells that have undergone necrotic cell death. This method, therefore, is not suitable for distinguishing between necrotic cell death and apoptotic cell death. (40, 41) In addition, this methodology does not permit the simultaneous detection of 3'-hydroxyl groups and other biologically relevant molecules, such as RNA and proteins.
Presently, there is a need in the art for a methodology to specifically detect apoptotic cells. There is presently no methodology that permits the specific detection of apoptotic cell death without the simultaneous detection of necrotic cells. In the experiments reported here, we determined whether DNA double strand breaks characteristic of those produced by an endonuclease like DNase I can be detected in apoptotic cells in situ.
When DNA is bound to histones or other proteins in chromatin, it is partially protected from the action of endonucleases, which are able to cleave the DNA at approximately 10-bp intervals, the distance of a single helical turn of the DNA (16). Because of the helical twist of DNA, the two strands are accessible to endonucleases with production of staggered ends as well as some blunt ends. Thus DNase I cleavage of nucleosome-bound DNA gives rise to double strand cuts with 1, 2, or 3 bases of 3' overhang (17, 18).
In contrast, DNase II cleavage of DNA in chromatin yields longer 3'-overhangs of an average of 4 bases (16, 18).